Gene expression in eukaryotes involves a complex series of RNA maturation events. Most aspects of these reactions are poorly understood in comparison to the analogous processes in prokaryotes because of the relative lack of sophisticated genetic approaches and a paucity of precursor RNAs with which to study the enzymatic reactions. Recently the use of highly efficient in vitro systems for the transcription of cloned tRNA genes has made it possible to synthesize both wild type and selectively mutagenized tRNA primary transcripts. These RNAs act as substrates for a variety of tRNA processing reactions in vitro, including nucleolytic maturation of the 5' terminus. Considerable attention has been focused on this last reaction in prokaryotes due to the fact that catalysis appears to be performed primarily by the RNA component of the enzyme (RNase P). In Saccharomyces cerevisiae this reaction is catalyzed by an endonuclease which also appears to be a protein moiety complexed with RNA. This activity will be purified from yeast nuclei, the enzymatic component(s) physically characterized and the sequence of any RNA moiety determined. In order to establish mechanisms for recognition and cleavage, structural analysis of the substrate and enzymic RNAs will be undertaken in conjunction with substrate utilization studies of selected mutants in the pre-tRNA substrates. These results will be compared with models for the analogous prokaryotic and mitochondrial reactions in order to establish a mechanism for the involvement of the RNA and ribonucleoprotein structures in nucleolytic catalysis.